Whooping cough remains an unconquered disease, worldwide. Developing nations simply do not have the resources to vaccinate their children. Very little disease is seen in the United States, and this is entirely due to the use of the whole-cell vaccine. However, this situation could change rapidly if the population refused to except the current whole cell vaccine, a situation which occurred in Great Britain and Sweden, where concern about the safety of this vaccine has lead to a decline of its use, and a subsequent rise in disease. The current whole cell vaccine contains only whole killed bacteria. A component vaccine of known composition would be desirable. The problem in developing a new component vaccine is to define what is necessary and sufficient to induce a protective immune response. We have focussed our efforts on trying to understand the disease, hoping to develop a theoretical basis for vaccine development. A unique aspect of the biology of Bordetella pertussis makes it an ideal model system to study pathogenesis. All of the genes required for virulence (perhaps 20), are coordinately expressed and regulated by the growth conditions. All are expressed at 37 degrees C (the temperature of the human host) but not at 25 degrees C. Also, high concentrations of MgSO4 can repress the synthesis of the virulence genes at 37 degrees C. We have shown that one gene (called vir for virulence-regulating) acts as a positive transcriptional inducer for the expression of the virulence genes. We propose to identify these gene products necessary for the virulent state and to study their role in the disease. Previously, mutants were identified using transposon Tn5 lac, a derivative of Tn5 which retains the transposition functions, kanamycin resistance, and a "reporter" gene, the gene for Beta- galactosidase (Beta-gal), without a functional promoter. Expression of Beta-gal occurs only when Tn5 lac has inserted into a functional gene. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified as kanamycin-resistant mutants that express Beta-gal when the vir-regulated genes are on, but not when the vir-regulated genes are off. In the current proposal we will continue to identify and characterize the virulence factors of Bordetella pertussis, both genetically and for their role in the disease.